The immune response of a vertebrate system provides a protective system that distinguishes foreign entities from entities native to the vertebrate system. Immune responses are the primary responsibilities of the B and T lymphocytes, which mediate the humoral response and the cell-mediated response, respectively. The humoral response is elicited by the B-cells which secrete antibodies (also known as immunoglobulins). Antibodies or immunoglobulins are molecules that recognize and bind to specific cognate antigens. Because of their exclusive specificities, antibodies, particularly monoclonal antibodies, are essential tools for analyzing the functions of biological molecules. Antibodies can be used to detect the protein expression levels, identify the protein-protein interaction complexes, localize the cellular compartment and tissue specificity, and analyze gene functions by neutralizing the gene product. Furthermore, antibodies have been widely used in the diagnosis and treatment of a variety of human diseases.
The basic immunoglobulin (Ig) in vertebrate systems is composed of two identical light (“L”) chain polypeptides (approximately 23 kDa), and two identical heavy (“H”) chain polypeptides (approximately 53 to 70 kDa). The four chains are joined by disulfide bonds in a “Y” configuration. At the base of the Y, the two H chains are bound by covalent disulfide linkages. The L and H chains are organized in a series of domains. The L chain has two domains, corresponding to the C region (“CL”) and the other to the V region (“VL”). The H chain has four domains, one corresponding to the V region (“VH”) and three domains (CH1, CH2 and CH3) in the C region. The antibody contains two arms (each arm being a Fab fragment), each of which has a VL and a VH region associated with each other. It is this pair of V regions (VL and VH) that differ, from one antibody to another (due to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen-binding site. More specifically, each V region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR). The CDR's are the most variable part of the variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation and selection.
Research in recent years has demonstrated that the function of a binding antigen can be performed by fragments of a whole antibody. Exemplary antigen binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the dAb fragment (Ward, E. S. et al., Nature 341, 544–546 (1989) which consists of a VH domain; (iv) isolated CDR regions; and (v) F(ab′)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (vi) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is the smallest functional unit required for high affinity binding of antigen.
One major challenge in the antibody field has been to reconstitute a vast diverse repertoire of immunoglobulins that mimics the immunoglobulin pool in the human immune system. Such a repertoire generally has a complexity ranging from 108 to 1013 distinct immunoglobulins. The generation of such a repertoire would greatly facilitate the identification and production of immunoglobulins capable of interacting specifically with therapeutic targets. However, the design and production of such a repertoire has traditionally been hampered by the lack of a stabilizing means for assembly of the minimal functional unit, namely the Fv fragment. It is a well-known problem in the art that the VH and VL regions, when expressed alone, have very low interaction energy (Glockshuber et al. (1990) Biochemistry 29(6):1362–1367). The two components dissociate at low protein concentrations and are too unstable for many applications at physiological body temperature. It is also a long-recognized technical obstacle that large proteins, such as whole antibodies (albeit extremely stable), do not express at an appreciable level in the host cell, thus rendering the construction of a highly diverse antibody repertoire very difficult.
More recently, three approaches have been developed to generate stable VL and VH complexes. However, each of these techniques bears a number of intrinsic limitations; and none of them circumvents the aforementioned technical hurdles completely. The first approach uses a peptide linker to connect the VL and VH as a single-chain (“scFv”) (Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A 85:5879–5883). While the resulting scFv exhibits substantial antigen-binding activity, not all antibodies can be made as single chains and still retain high binding affinity (Huston et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879–5883; Stemmer et al. (1993) Biotechniques 14(2): 256–265). In part, this is due to the interference of linker sequences with the antigen binding sites. Furthermore, the propensity of single-chain antigen-binding units to aggregate inside a cell also hampers their intracellular antigen-binding capabilities. To efficiently isolate those single-chain antigen-binding units with the desired intracellular binding capabilities, a vast diverse repertoire of distinct single-chain antibody molecules that are amenable for an in vivo selection must be generated.
The second approach involves inserting a pair of cysteine residues in the VL and VH regions to generate a disulfide-bond stabilized Fv (“dsFv”) (Brinkmann et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90(16): 7538–7542). The incorporated disulfide linkage, however, is unstable under reducing conditions in many host cells. For instance, in cytosol of E. Coli, the inter-molecular disulfide bond is often insufficient to stabilize the VL and VH complex. Moreover, this method typically requires 3-dimensional structural information of the V regions to ensure that the cysteine pair is inserted in a proper place without disruption the binding activity. Because the 3-dimensional information of a vast majority of the existing antibodies is unknown, this approach has little practical utility, and is particularly unsuited for antibody library construction, especially for constructing antibody repertoires derived from B cells. The third approach for stabilizing the VL and VH regions utilizes the disulfide bonds native to the CH1 and CL domains. This method proceeds with grafting a disulfide-bond linked CH1 and CL domains to the C-termini of the VL and VH regions in order to reconstitute a Fab fragment. While the resulting Fab fragment is generally more stable and often exhibits higher binding affinity than scFv, Fab is not optimal for high level expression and antibody repertoire construction due to its large size.
Certain dimerization sequences that form coiled-coil structures have also been employed to assemble multivalent antibodies. Specifically, U.S. Pat. No. 5,932,448 describes a bispecific F(ab′)2 heterodimer linked by the Fos and Jun leucine zippers. However, the binding sites are still stabilized by the constant regions (e.g. CH1) contained in the F(ab′)2 molecule.
Thus, there remains a considerable need for improved compositions and methods to generate stable antigen-binding units and repertoires thereof to effect identification of therapeutic antigen-binding units. An improved antigen-binding unit would be more stable than a Fv fragment, but would preferably be smaller than a Fab fragment to allow large-scale production and efficient display. Such antigen-binding unit would also serve as a building block for constructing multivalent and/or multispecific antibodies. The present invention satisfies these needs and provides related advantages as well.